Background: Few studies describe the community-acquired pneumonia (CAP) burden in children in Asia. We estimated the proportion of all CAP hospitalizations in children from nine hospitals across the Republic of Korea (high-income), Indonesia, Malaysia (middle-income), and Vietnam (low/middle-income).Methods: Over a one or two-year period, children <5 years hospitalized with CAP were identified using ICD-10 discharge codes. Cases were matched to standardized definitions of suspected (S-CAP), confirmed (C-CAP), or bacterial CAP (B-CAP) used in a pneumococcal conjugate vaccine efficacy study (COMPAS). Median total direct medical costs of CAP-related hospitalizations were calculated.Results:Vietnam (three centers): 7591 CAP episodes were identified with 4.3%  S-CAP, 3.3%  C-CAP and 1.4%  B-CAP episodes of all-cause hospitalization in children aged <5 years. The B-CAP case fatality rate (CFR) was 1.3%. Malaysia (two centers): 1027 CAP episodes were identified with 2.7% ; 2.6% ; 0.04%  due to S-CAP, C-CAP, and B-CAP, respectively. One child with B-CAP died. Indonesia (one center): 960 CAP episodes identified with 18.0% ; 16.8% ; 0.3%  due to S-CAP, C-CAP, and B-CAP, respectively. The B-CAP CFR was 20%. Korea (three centers): 3151 CAP episodes were identified with 21.1% ; 11.8% ; 2.4%  due to S-CAP, C-CAP, and B-CAP, respectively. There were no deaths.Costs: CAP-related hospitalization costs were highest for B-CAP episodes: 145.00 (Vietnam) to 1013.3 USD (Korea) per episode.Conclusion: CAP hospitalization causes an important health and cost burden in all four countries studied (NMRR-12-50-10793). Pneumonia is the leading cause of childhood deaths worldwide.Streptococcus pneumoniae (S. pneumoniae) causes around one-third of childhood deaths due to pneumonia.S. pneumoniae.The lack of a ‘gold standard’ in diagnosing pneumonia and establishing its etiology remains a major obstacle in determining the burden of pneumonia due to individual pathogens.The introduction of PCVs into routine childhood immunization schedules is one of several key measures identified by WHO to achieve a global reduction in childhood pneumonia.Synflorix, GSK, Belgium) according to a 3-dose primary vaccination schedule, showed positive vaccine efficacy against CAP.Data from the randomized controlled Clinical Otitis Media and Pneumonia Study (COMPAS), in which more than 10,000 children from middle-income countries in Latin America received the 10-valent PCV  episodes were identified in three study hospitals in Hue City, Hanoi and Ho Chi Minh City; 4,571 (75.7%) could be classified as confirmed CAP (C-CAP) and 1,542 (25.5%) as B-CAP , 8.3/1,000 person-years , and 0.4/1,000 person-years , respectively. Assuming that 25.5% of episodes with absent CXR (n = 540) in Hue City were B-CAP episodes (n = 138), then the incidence of B-CAP was 6.1/1,000 person-years . There was a seasonal trend in CAP hospitalizations (any category) with two peaks; one in March and one in September.Haemophilus influenzae type b (Hib) or influenza vaccine (Sup Table 1).Of 1,542 recorded B-CAP episodes identified in the three hospitals, 20 resulted in death . In children with B-CAP, 749 (48.6%) recovered with sequelae . The higThe median duration of hospitalization was 7 days (range: 1–66 days) across all categories of CAP. It was longest in infants ≤6 weeks of age and children 48–59 months of age  and shortest in 24–35 month olds .The total median direct medical cost of B-CAP-related hospitalization was 145.00 USD, per episode .Table 2Applying the vaccine efficacy results from the COMPAS trialUsing incidence rates from Hue City, we estimated the overall annual hospitalized CAP burden in Vietnam to be 85,179 cases of hospitalized S-CAP; 60,436 cases of C-CAP and up to 44,597 cases of B-CAP. Under the different scenarios described above, a vaccination program with PHiD-CV in Vietnam could potentially have prevented between 5,789 and 9,811 B-CAP hospitalizations nationwide during the one-year study period.From December 2009 to December 2011, 1,004 hospitalized S-CAP episodes were identified in the two study hospitals in the cities of Serembam and of Kota Kinabalu; 957 (95.3%) of these could be classified as C-CAP and 15 (1.5%) as B-CAP episodes . A furthOf 15 B-CAP episodes, 12 children fully recovered and one died . The higThe median duration of hospitalization was longest for B-CAP episodes  and shortest for S-CAP and C-CAP . Hospitalizations were longest in infants aged ≤6 weeks  and shortest in age groups above 24 months .The total median direct medical cost of B-CAP-related hospitalization was 375.80 USD, per episode .From January 2010 to November 2011, 949 hospitalized S-CAP episodes were identified in a hospital in the city of Bandung, 886 (93.4%) of which could be classified as C-CAP, and 15 (1.6%) as B-CAP . Only 7.S-CAP episodes represented 18.0%  ; C-CAP episodes 16.8%   and B-CAP episodes 0.3%   of all hospitalizations in children aged <5 years. The proportion of S-CAP hospitalizations  was highest in children 7 weeks-12 months of age: 38.5% . There were 873 children (96.2%) who had one S-CAP episode. The number of CAP episodes peaked in March in each study year.Among 15 B-CAP episodes, eight children (53.3%) fully recovered and three died (20%) . There wThe duration of hospitalization was longest for B-CAP episodes (median 10 days range 4–46) and shortest for S-CAP and C-CAP episodes . Among S-CAP episodes, the duration of hospitalization was longest in infants aged ≤6 weeks (median 8 days range 2–25) and shortest in 48–59 month-olds . The majority of S-CAP episodes (63.1%) were in children between 7 weeks-12 months of age .The total median direct medical cost of B-CAP-related hospitalization was 200.30 USD, per episode .From January to December 2011, 2,962 hospitalized S-CAP episodes were identified in the three study hospitals, two located in Seoul and one in the city of Wonju, of which 1,656 (55.9%) were C-CAP and 335 (11.3%) were B-CAP episodes . A furthThe majority of children (93.8%) had one CAP episode over the study period. The number of CAP episodes peaked in May and September to November. More than 99% of CAP episodes  recovered and there were no deaths .The highest proportion of CAP admissions was in children aged 7 weeks-12 months and 13–23 months . Among B-CAP episodes, 51.9% were in male children, a co-morbid condition was present in 0.9% of episodes, and approximately 80% reported having received at least one dose of PCV or Hib vaccine (Sup Table 3).The median duration of hospitalization was 4 days for S-CAP (range 1–141 days) and 5 days for C-CAP (range 1–38 days) and B-CAP (range 2–38 days), and was similar (median 4 or 5 days) in each age category.The total median direct medical cost of B-CAP-related hospitalization was 1,013.30 USD per episode . More thHospital discharge databases were used to estimate the burden of hospitalized S-CAP, C-CAP and B-CAP among children <5 years of age in four countries in the Southeast and Western Pacific Regions of Asia. The four countries differ in terms of their healthcare systems , vaccination schedules and income status. Thus, while the CAP disease burden and associated direct medical costs were substantial in all four countries, observed differences in results are to be expected.CAP hospitalizations as a proportion of all hospitalizations were highest in Korea  and Indonesia (a middle income country with some areas of poverty), suggesting high incidence and/or low threshold for hospitalization in these countries. The proportion of CAP hospitalizations classifiable as B-CAP  ranged from 1.5–25.5% across the countries, representing between <1%  and 1.4% (Vietnam) of all-cause hospitalizations in children aged <5 years during the study period. Elevated CRP was a diagnostic criteria for B-CAP in our study, yet CRP was tested in 64.0% of total CAP cases in Vietnam and in 7.8% or fewer cases in Indonesia and Malaysia (versus 99% in Korea), suggesting that B-CAP cases may have been significantly underestimated in these countries. In all countries, a higher percentage of hospitalized CAP episodes  were in children aged <24 months. However, the proportion of episodes in younger versus older children showed the least variability in Korea.There were more deaths in children with B-CAP than with S-CAP or C-CAP, with the highest CFR rate observed in Indonesia; no deaths were recorded in Korea. A weight-for-age Z-score <-2 (moderate or severe underweight for age) has been linked to an increased risk of childhood pneumonia.We were able to estimate the CAP incidence rate in children <5 years of age in Hue City, Vietnam for 2011. The incidence of B-CAP at this center is, in all probability, an underestimate due to the large number of unclassifiable CAP episodes : indeed B-CAP as a proportion of S-CAP was much lower in this center  than the other Vietnamese centers . Thus, the adjusted incidence rate of 6.1/1,000 person-years may be more likely to be nationally representative of B-CAP hospitalizations in children in Vietnam. However, our adjusted B-CAP estimate is higher than the results from a prospective population-based study in Nha Trang city, Vietnam in 2006, where the incidence of radiologically-confirmed hospitalized CAP in children aged <5 years was 3.3/1,000.Due to the limited number of studies describing CAP burden in children in Asia, their slightly different definitions and patient recruitment from inpatient and/or outpatient settings, it is difficult to compare these findings with our observations. Nevertheless, the incidence of S-CAP in Hue city  is comparable to estimates for Southeast Asia  and the Western Pacific  in a recent literature review of severe  acute lower respiratory tract infection (LRTI) in children aged <5 years.Assuming PHiD-CV vaccine efficacy of 22.0% against B-CAP, PHiD-CV vaccination would have been expected to prevent up to 425.7 B-CAP hospitalizations, 5.5 deaths, and 61,726 USD over one year in the Vietnamese study hospitals. By extrapolating data from Hue City to the whole of Vietnam, we estimate that PHiD-CV vaccination could potentially have prevented between 5,789 and 9,811 B-CAP hospitalizations nationwide in 2011, or up to 1,422,644 USD.S. pneumoniae.There were few B-CAP episodes  in the middle-income countries of Malaysia and Indonesia, probably reflecting the very low rates of CRP testing done in these centers. In the absence of denominator data  we were unable to estimate CAP incidence rates, limiting comparisons to be made with other studies. However, some extrapolation and comparison of data between studies were possible. Aljunid et al, by using data from six hospitals in Malaysia, estimated that the incidence of pneumonia in children was 7.6/1,000 for all-cause pneumonia and 2.4/1,000 for pneumococcal pneumonia (2006-2007).Data from other middle-income countries also indirectly suggest that our study may have underestimated the B-CAP burden in Malaysia and Indonesia: In a large population-based study (2005-2010) the incidence of hospitalized acute LRTI in children <5 years of age in Thailand was estimated to be 57.7/1,000 person-years, with a CFR of 0.3%.PCVs were not included in the national immunization programs (NIP) of any of countries at the time of the study, but were available in the private market. Approximately 78% of children in Korea had received at least one PCV dose, which is in line with published data (>74% of the 2010 Korean birth cohort received the PCV primary series).Very small proportions (<1%) of children in Vietnam and Indonesia had received Hib vaccination, compared with over 75% in Malaysia and Korea. Hib-containing vaccines were included in the Malaysian NIP in 2002, Vietnam in June 2010, and not until after our study in Korea (2013) and Indonesia (2014). The unexpectedly low Hib vaccination rates reported in Vietnam may reflect incomplete recording of Hib vaccination in hospital medical records.The disease CAP was associated with a significant cost burden in all countries. The highest median direct medical costs in all study sites were associated with B-CAP hospitalizations. Due to the potential differences in the case-definition, sample size and geographic locations, we were not able to make direct comparisons of the cost data from our study with those reported in the literature from different countries. However, we have noted that our results were comparable with, or within a reasonable range of the data reported in published studies from Vietnam, Malaysia and Korea.The strengths of our study include the large number of medical records reviewed for CAP episodes; the use of a single harmonized protocol between the four countries which allowed application of the same CAP definitions, data collection methods, and statistical analysis; the geographical distribution of sites in Vietnam, Korea and Malaysia offering good representation of the countries studied; the availability of one site where incidence could be estimated; and the collection of cost data providing additional insight into the CAP burden in the four countries. Nevertheless, in view of differences in health care systems and potential differences in disease management, health care seeking behaviors, and ICD 10th Revision (ICD-10) coding procedures between individual countries, comparisons across countries should be avoided and our study may have limited applicability to other regions. Even if the 4 countries may differ in terms of healthcare and delivery systems and healthcare seeking behaviors, they are part of Asia and the overall results of this study suggest that independently of the health care system considered, there is a significant number of hospitalizations attributable to community acquired pneumonia  and there is an appreciable burden of healthcare costs associated with these episodes of hospitalizations .The single study center in Indonesia means any extrapolation to the national level should be undertaken cautiously, while the predominately urban locations studied potentially limit the generalizability to rural populations. Our study focused on hospitalized cases, but in low/middle- and middle-income countries many pneumonia cases may not access hospital care, and more than 80% of deaths may occur outside hospital.S. pneumoniae serotypes were published from the population covered by the study sites in Korea at the time of the protocol development of this study, as well as also the catchment population of the hospital sites in Korea could not be defined. Finally, the analysis of cost data was limited to a subset of validated episodes in Vietnam and Malaysia. Only direct medical costs were assessed in the study, which only provides a fraction of the total costs associated with pediatric pneumonia hospitalizations.There was a lack of CRP testing, which precluded a diagnosis of B-CAP in most children in Indonesia and Malaysia, and another potential limitation of the study was that the study duration in Vietnam and in the Republic of Korea could only last for one year and this because of logistical difficulties. We applied a retrospective design relying on individual physicians' diagnosis of pneumonia (and ICD-10 coding) in each of the centers. Therefore no uniform diagnostic criteria for pneumonia were applied. We were unable to assess any impact of coding misclassification in any of the centers. We evaluated CXR results using capture words  and did not conduct an expert review, which may have over- or under-estimated the proportion of B-CAP episodes identified. We decided to extrapolate the PHiD-CV efficacy for prevention of CAP from COMPAS to Vietnam only, as the CRP laboratory test was not widely used in Malaysia and Indonesia to classify the cases. No data on the circulating This is one of few studies to assess the burden of hospitalized CAP in children living in the Western Pacific and Southeast Asia region. While there are apparently marked differences between countries in terms of the CAP burden and the characteristics of children who are admitted to hospital with CAP, the study indicates an important CAP burden in all of the countries studied. The few published studies estimating the pneumonia burden in the region differ in terms of setting, age group studied, and CAP case definition used, making comparisons between studies challenging. There is also a need for surveillance studies to be conducted in Malaysia, Vietnam, and Indonesia that are based on active, prospective enrolment of children suffering from CAP in hospital and community sites. Ideally these studies should be conducted in defined populations to allow accurate estimates of incidence to be made. Alternatively, studies to validate the performance of the coded discharge diagnoses in these countries, in order to accurately and completely identify all CAP cases in children, would provide valuable information.Vaccination is identified as one of the key interventions in pneumonia prevention, that could reduce the observed health inequalities between countries, and reduce morbidity and mortality due to CAP.The primary study objective was to estimate the proportion of all hospitalizations in children aged <5 years that could be attributed to CAP . The clinical outcome of CAP episodes, duration of CAP hospitalizations, and the cost burden were also assessed. The incidence of hospitalization due to CAP was determined for Hue City Vietnam, which had a defined hospital catchment area. The study period lasted 1 year (January-December 2011) in Vietnam and Korea, but lasted 2 years (January 2010-December 2011) in Indonesia and Malaysia. For Vietnam, where incidence rates could be calculated, the number of hospitalizations, deaths, and costs associated with B-CAP, and potentially preventable by vaccination with PHiD-CV were estimated by applying the COMPAS trial resultsThe study was conducted according to the Declaration of Helsinki and Good Clinical Practice guidelines. The study protocol and associated documents were reviewed and approved by institutional review boards in each participating country. All data were anonymized and informed consent was not required.The study was conducted in nine hospitals in four countries: three referral hospitals in Vietnam ; two referral hospitals in Malaysia ; one tertiary referral hospital in Indonesia ; and three referral hospitals in Korea .The coverage of routine pediatric vaccines varies in Vietnam , but is consistently high in Indonesia, Malaysia and Korea (range 86% to >99% for DTP3) (2013 data).The four studied countries are economically diverse. The World Bank classifies Vietnam as a low/middle-income country, Malaysia as an upper middle-income country, Indonesia as a lower middle-income country, and Korea as a high-income country.S. pneumoniae, pneumonia due to Hib, unspecified bacterial pneumonia and pneumonia-organism unspecified; presented in supplemental digital content). Medical records were selected on the basis of first and any listed ICD-10 codes relating to pneumonia. Information on demographics, duration of hospitalization, outcome of hospitalization, medical history , nutritional status, vaccination history  and the presence of any comorbid conditions  were extracted from medical records. CRP levels and results from CXR were also retrieved from medical records in order to categorize CAP in the analysis.Medical records of participating hospitals were reviewed to identify children aged <5 years with diagnosed CAP, using the ICD-10 codes J12-J22, J85-J86  and itemized costs of the corresponding resource were retrieved from the hospital database and Ministry of Health website respectively, to compute the total costs.http://www.huecity.gov.vn) and used as the denominator. The total population of children <5 years of age in Vietnam was estimated from United Nations statistics for 2010.For incidence estimation, all enrolled subjects with ‘Hue’ as their city of residence were included for the numerator. Data on the total number of children aged <5 years living in Hue City in 2011 were obtained from the official Hue City website  due to CAP among all hospitalizations in children aged <5 years was estimated using the total number of hospitalizations during the study period as the denominator. The proportion of S-CAP, C-CAP and B-CAP cases was estimated by age group , gender, month and year of hospitalization. Weight-for-age z-scores were computed using WHO criteriaThe incidence (and exact 95% CI) of CAP episodes in hospitalized children in Hue City, Vietnam was calculated by CAP status. The number of hospitalizations and deaths from B-CAP potentially prevented, and costs potentially saved by vaccination with PHiD-CV in Vietnam were estimated by applying the vaccine efficacy results from COMPAShttp://www.oanda.com/currency/converter/ in December 2013).Median total direct medical costs of CAP-related hospitalizations were calculated per country and adjusted to 2011 values using the Consumer Price Index of the country, wherever applicable. In Vietnam, cost data were analyzed for a 10% random sample taken across all three centers (n = 595). In Malaysia, cost data were analyzed for 20% of randomly selected CAP episodes from one center and for all episodes from the other center (n = 349). In Korea, unsubsidized total direct medical costs, associated costs for reimbursable items (government subsidy and patient out-of-pocket amounts) and non-reimbursable items were collected from hospital administrative databases from all three centers. Costs were presented in USD  as a proxy for pneumonia or CAP.SAS Version 9.2 with Drug and Development web portal version 3.5.The statistical analysis used


The expressions of PKR-like ER kinase (PERK) and CCAAT/enhancer-binding protein-homologous protein (CHOP) were detected by immunohistochemistry. The cell apoptotic rates of AECs were analyzed by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) staining. The expression levels of light chain 3 (LC3-II), p62, Beclin-1, ATG5, ATG7, Caspase-12, and Caspase-3 were detected by Western blotting. Results showed that the COPD group exhibited a lower FEV0.3/FVC% and Cdyn, and a higher RI than the control group. Compared with the control group, the integrated optical density (IOD) values of PERK and CHOP, the apoptotic rate of AECs, and expressions of LC3-II, Beclin-1, ATG5, ATG7, Caspase-3, and Caspase-12 expressions were significantly higher, whereas p62 expression was significantly lower in the COPD group. Based on the results obtained during the present study, it became clear that the inhibition of autophagy could attenuate the ERS-induced apoptosis of AECs in rats with COPD.During the present study, we explored the protective effects of autophagy on endoplasmic reticulum (ER) stress (ERS) induced apoptosis belonging to alveolar epithelial cells (AECs) in rat models with chronic obstructive pulmonary disease (COPD). Fifty-six 12-week-old male Sprague–Dawley (SD) rats were randomly assigned into the COPD group (rats exposed to cigarette smoke (CS)), the 3-methyladenine (3-MA) intervention group (COPD rats were administrated with 10 mg/kg autophagy inhibitors), the chloroquine (CQ)-intervention group (COPD rats were administrated 40 mg/kg CQ), and the control group . The forced expiratory volume in 0.3 s/forced vital capacity (FEV Chronic obstructive pulmonary disease (COPD) is a term used to describe a group of diseases that previously included bronchitis and emphysema . COPR isER is a unique organelle for protein synthesis, folding, and delivery in the cell and it is essential in numerous cellular functions . An imbath day and from the 15th to the 28th day. Rats in the 3-MA intervention group were used to establish the rat model of COPD, and were intraperitoneally injected with 3-MA (10 mg/kg), 0.5 h before exposure to CS and intratracheal instillation of LPS. Rats in the CQ intervention group were intraperitoneally injected with 3-MA (40 mg/kg), 0.5 h before exposure to CS and intratracheal instillation of LPS except for preparing the COPD model. Rats in the control group received intratracheal instillation of sterilized normal saline on the 1st and the 14th day. From the 2nd to the 13th day and from the 15th to the 28th day, rats were left to breathe in a glass box with normal air for the same period of time with rats in the other groups. General conditions and statuses of all rats were observed everyday, including their diet, activities, cough, and other general conditions, and they were fed a normal diet for 4 weeks before further experiments.A total of 56 specific pathogen-free (SPF) male Sprague–Dawley (SD) rats that were 12 weeks old and weighed between 250 and 280 g were used in the present study. Rats were initially allowed to acclimate for 1 week. All the rats were randomly assigned and evenly divided into four experimental groups with 14 rats in each group: the control group, the COPD group, the chloroquine (CQ) intervention group, and the 3-methyladenine  intervention group. Rats in the COPD group were anesthetized on the 1st and the 14th day, and an incision was made to expose the trachea for intratracheal instillation of lipopolysaccharide  using a 4-gauge needle. Rats were then exposed to CS produced by 12 filter-tipped Furongwang cigarettes obtained from the Cigarette Factory . Exposure to CS was performed in a 72-l closed glass box for 0.5 h everyday, starting from the 2nd to the 130.3/FVC%), inspiratory resistance (RI), and dynamic lung compliance (Cdyn) were calculated by Maclab data acquisition system.Anesthetized rats in each group were positioned in the supine position on the operation table, and an incision was made in the anterior part of the neck in order to expose the trachea and insert a tracheal intubation tube. The other end of tracheal intubation was connected to a pressure sensor, a flow sensor, a ventilator, and Maclab data acquisition system . The tidal volume was set at 10 ml/kg, and the respiratory rate was set at 60 breaths/min. The breathing pattern of the rats was observed for a period of time, after which air was rapidly inflated into the trachea with a volume of five times the tidal volume. The ventilator was then immediately disassembled and replaced with an aspirator of negative pressure for air removal. Afterward, the forced expiratory volume in 0.3 s/forced vital capacity . The abdominal cavity was fully exposed to allow access into the thoracic cavity with an abdominal midline incision, avoiding lung or heart injury. The self-made rat lung lavage device (a 10-ml injector with 27-gauge needle) was inserted into the left ventricular apex of rats. The lavage was performed for approximately 2 min using PBS which was passed into the aorta from the left ventricle of heart and circulated throughout the body. When the pink lung tissues gradually turned pale and off-white, the left and right lung tissues were incised from the rats by rapid exposure of the trachea and lung. Portions of removed lung tissues were stored in liquid nitrogen, the rest of which was immersed in 4% paraformaldehyde and embedded with paraffin. Tissues were then sliced into successive sections (4-μm thick). These sections were preserved for later Hematoxylin–Eosin (HE) staining and immunohistochemistry. Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) staining was applied to detect the apoptotic rates of AECs.Three lung tissue sections were randomly selected from each group. All sections were dewaxed with xylene and hydrated with ethanol. Sections were stained by Hematoxylin, and differentiated by hydrochloric acid-ethanol solution. Next, they were counterstained by Eosin, and finally dehydrated by ethanol. The samples were observed under light microscope after being dewaxed with xylene and sealed with neutral gum.The expression of PKR-like ER kinase (PERK) and CHOP proteins in lung tissues were measured by immunohistochemistry. Lung tissues were first sliced into sections, and then dewaxed with xylene and dehydrated by alcohol. The activity of endogenous peroxidase was blocked with the addition of 3% hydrogen peroxide. Then, the slices were transferred to 0.01 mol/l of citrate buffer solution (pH 6.0), and heated in a microwave oven to retrieve antigens for 20 min. PBS rinsing was conducted three times after natural cooling (5 min per rinse). After antigen retrieval, rabbit-anti-mouse PERK or CHOP monoclonal antibody , were added to the tissue sections and incubated at 37°C for 1 h. Tissue sections were then incubated at 37°C for another 30 min with the second antibody, biotinylated goat anti-rabbit IgG. Streptavidin-labeled horseradish peroxidase (S-A/HRP) was added to the tissues and was left to further incubate for 10–15 min at 37°C. Tissue sections were stained with diaminobenzidine (DAB) for 1–2 min, and washed with PBS three times (2 min per time). Tissue sections were counterstained by Hematoxylin for 1 min, dehydrated by alcohol, and sealed with neutral gum. The positive sections and PBS that replaced the primary antibody served as the positive control and negative control. After staining, the positive tissue sections appeared as brown or tawny-brown when observed under the microscope. The integrated optical density (IOD) values of tissue sections in each group were measured by Image-Pro Plus 6.0 software after tissue images were captured under an optical microscope (×400). Five views were randomly selected to determine the positive IOD values, and the mean IOD values were considered as the relative expressions of PERK and CHOP.The apoptotic rates of AECs in rats were detected using a TUNEL detection kit . The lung tissues of rats in all the three groups were fixed with 10% paraformaldehyde at room temperature for 30 min, and then incubated in an ice-bath with PBS containing 0.1% Triton X-100 for 2 min. After allowing to dry, 50 μl of the TUNEL reaction mixture (the ratio of enzyme solution and label solution was 1:9) was added to samples, and left to incubate in the wet box at 37°C for 60 min. Following a PBS wash, 50 μl of transforming agent was added to tissue samples, after which tissues were incubated in the wet box at 37°C for another 30 min. Tissue samples were washed with PBS, followed by coloration with 100 μl of DAB substrate solution. And 10 min later, tissues were observed under a light microscope. Yellow-brown granules observed in the nucleus of rats indicated TUNEL-positive cells. Three visual fields were randomly selected from each tissue section using a microscope, in which apoptotic AECs were counted and the mean apoptosis rate was calculated. Apoptotic rate was calculated as the number of apoptotic AECs divided by the number of total AECs.Proteins were extracted from lung tissues of rats in each group. The BCA assay  was performed to detect the total concentration of the proteins. SDS/PAGE  was performed to separate proteins. Separated proteins were transferred on to PVDF membrane, which was blocked with 5% skim milk and left on the shaker for 1 h at 37°C. Later, the proteins were treated with the primary antibodies against light chain 3 (LC3-II), Caspase-12, and β-actin , rabbit-anti-rat p62 , rabbit-anti-rat Beclin-1 , rabbit-anti-rat ATG5 , rabbit-anti-rat ATG7 , and rabbit-anti-rat Caspase-3  at 4°C overnight. The membrane was then washed with TBS-Tween 20 (TBST). Protein samples were incubated for another 2 h with HRP-labeled goat anti-rabbit IgG . After the membrane was washed, the proteins were developed with chemiluminescence reagent, and gray-scale value of protein bands were measured by ImageJ software. Formula: the relative expression of the target protein was calculated by dividing the gray-scale value of target band by the gray-scale value of internal reference band. Each experiment was repeated three times.ttest. All the statistics were bilaterally calculated with the significant test level of α =0.05. Values of P<0.05 were considered as statistically significant.All experimentally obtained data were analyzed by SPSS 21.0 statistical software . Measurement data were presented as a mean ± S.D., and comparison of data between two groups was conducted using a The wounds of all the rats healed almost completely within a week after receiving intratracheal instillation of LPS or sterilized normal saline for the first time. When the rats underwent intratracheal instillation of LPS for the second time, rats in the control, CQ intervention, and 3-MA intervention groups recovered well. The healing rate of rats in the COPD group was slower than those after their first-time instillation. From the 15th to 28th day, rats in the control group retained a normal diet and appetite, sensitive response, and shiny skin, with a steady weight increase. In the COPD group, the rats exhibited a reduced diet intake, slow response, dry hair, and constant loss of the body weight. They also exhibited a rapid shallow and breathing in usual, and whoops under exposure to CS, accompanied with obvious paradoxical respiratory. The general conditions of rats in the 3-MA intervention and CQ intervention groups were worse than that in the control group but better than that in the COPD group.0.3/FVC% and Cdyn were significantly lower and RI was significantly higher in the COPD group compared with the control group . This indicates that the conditions and statuses of the rats (airflow obstruction and obstructive ventilatory dysfunction) were consistent with the changes in pulmonary function in the rat models of COPD. In the 3-MA intervention and CQ intervention groups, the FEV0.3/FVC% and Cdyn were significantly higher than those in the COPD group, while RI was lower than that in the COPD group  .Pulmonary function tests showed that the FEV P<0.05) . No signImages of rat lung tissues stained with HE were shown as P<0.05). In the 3-MA intervention and CQ intervention groups, the IOD values of PERK and CHOP of lung tissues were significantly lower than those in the COPD group (both P<0.05). No significant difference was observed in the PERK and CHOP IOD values between the control and 3-MA groups (both P>0.05). These results indicated that the ERS was enhanced in the COPD group, which might be reduced by 3-MA intervention.The PERK positive expression dyed in the cytoplasm and the CHOP-positive expression dyed in the nucleus was visible in both BECs and AECs . The IODP<0.05). On the other hand, the apoptosis rate of AECs was markedly lower in the 3-MA intervention and CQ intervention groups compared with that in the COPD group (P<0.05). There was no significant difference in the apoptotic rates between the control and 3-MA intervention groups (P>0.05) (TUNEL staining demonstrated that the apoptotic rate of AECs in the COPD group were significantly higher than those in the control group ((P>0.05) .P<0.05). The 3-MA intervention and CQ intervention groups exhibited significant expressions of LC3-II, Beclin-1, ATG5, ATG7, Caspase12 and Caspase-3, and higher p62 expression in lung tissues compared with that of the COPD group (both P<0.05). No significant difference was found in the apoptotic related proteins expressions amongst the control, CQ intervention, and 3-MA intervention groups (both P>0.05).Western blotting was conducted to measure the expressions of apoptosis-related proteins in rats with COPD . CompareIn the present study, we investigated how the apoptosis of AECs induced by ERS in COPD rats as well as the protective effects of autophagy, can prove that the inhibition of autophagy itself might alleviate ERS-induced apoptosis of AECs in rat models of COPD. Autophagy is a natural cellular process during which protein and organelles are degraded in order to maintain cell viability and activity in response to nutrient limitation, and functions as a form cytoprotection and cell apoptosis ,21. Auto0.3/FVC% and Cdyn decreased while the RI was increased in the COPD group compared with the control group. In the present study, the rat models of COPD were successfully established by CS inhalation. CS exposure leads to early neurogenic inflammation in the airways through stimulating sensory neurones causing CS-evoked diseases, such as asthma or COPD [0.3/FVC% ratio represents an inhibited pulmonary ventilation function, and the decreased Cdyn indicates low lung compliance with an increasing intrapleural pressure [0.3/FVC% ratio and Cdyn significantly increased, whereby RI decreased in the 3-MA intervention and CQ intervention groups compared with that in the COPD group. Our results also indicated that the inhibition of autophagy can reduce bronchial wall thickening and pulmonary bullae in COPD rats. These results provided evidence that the inhibition of autophagy might improve pulmonary function in rats with COPD.Our study demonstrated that both 3-MA and CQ are beneficial in inhibiting the process of autophagy. 3-MA belongs to a group of phosphoinositide-3-kinase (PI3K) inhibitors, which have been widely used as autophagy inhibitors based on their inhibitory effects on class 3 PI3K activity, an essential component for the induction of autophagy . Petiot  or COPD . The sig or COPD . Consist or COPD . The decpressure . Airflowpressure . Furtherpressure . Bullae pressure . Interesin vitro, and when overexpressed in cells, can lead to the attenuation of protein synthesis [Sequentially, expressions of both PERK and CHOP in the COPD rats significantly increased. However PERK and CHOP expression levels both declined when autophagy was inhibited. The ERS is associated with specific proteins, such as PERK, CHOP, ATF4, GRP78, p-eIF2α, and Caspase12, whereby each of these proteins are closely associated with one another . PERK isynthesis . The traynthesis ,37. It iynthesis . CHOP exynthesis . Caspaseynthesis . Autophaynthesis ,42. Chenynthesis  suggesteynthesis  also repIn conclusion, our study mainly elucidated that the inhibition of autophagy improves pulmonary function and attenuates ERS-evoked AECs apoptosis in COPD rats exposed to CS. Therefore, the study supported that long-term 3-MA treatment might be a new therapeutic strategy for COPD. However, as a preliminary study, the underlying mechanism still needs to be validated by additional research with further attention paid to the regulation of cigarette smoking, inflammation, and reduction in ERS.


In this work, we estimate the radiation dosimetry for this ligand labeled with the alpha-emitter 68Ga-PSMA-617. Source organs were kidneys, liver, spleen, salivary glands, bladder, red marrow and representative tumor lesions. The imaging nuclide 68Ga was extrapolated to the half-life of 213Bi. The residence times of 213Bi were forwarded to the instable daughter nuclides. OLINDA was used for dosimetry calculation. Results are discussed in comparison to literature data for 225Ac-PSMA-617.Three patients with metastatic prostate cancer underwent PET scans 0.1 h, 1 h, 2 h, 3 h, 4 h and 5 h after injection of RBE5/GBq for salivary glands, 8.1 Sv RBE5/GBq for kidneys and 0.52 Sv RBE5/GBq for red marrow. Liver (1.2 Sv RBE5/GBq), spleen (1.4 Sv RBE5/GBq), bladder (0.28 Sv RBE5/GBq) and other organs (0.26 SvRBE5/GBq) were not dose-limiting. The effective dose is 0.56 Sv RBE5/GBq. Tumor lesions were in the range 3.2–9.0 SvRBE5/GBq (median 7.6 SvRBE5/GBq). Kidneys would limit the cumulative treatment activity to 3.7 GBq; red marrow might limit the maximum single fraction to 2 GBq. Despite promising results, the therapeutic index was inferior compared to 225Ac-PSMA-617.Assuming a relative biological effectiveness of 5 for alpha radiation, the dosimetry estimate revealed equivalent doses of mean 8.1 Sv 213Bi-PSMA-617 is in a range traditionally considered reasonable for clinical application. Nevertheless, compared to 225Ac-PSMA-617, it suffers from higher perfusion-dependent off-target radiation and a longer biological half-life of PSMA-617 in dose-limiting organs than the physical half-life of 213Bi, rendering this nuclide as a second choice radiolabel for targeted alpha therapy of prostate cancer.Dosimetry of The online version of this article (10.1007/s00259-017-3817-y) contains supplementary material, which is available to authorized users. Then, the decay formula was applied vice versa with T1/2 46 min from time of injection to the respective time point to calculate the activity of 213Bi. Thus, TACs intended to represent 213Bi-PSMA-617 were calculated, taking into account the different half-life of the modeled radionuclide but assuming that the nuclide replacement does not affect the pharmacokinetics of the shuttle molecule. Red marrow, total body, urinary bladder content and all segmented organ TACs . All six imaging time points were coregistered with automatic rigid and, if applicable, deformable coregistration of CT images. PET scans were coregistered by coupling to the transformation matrix of the coupled CT. Segmentation was done by a radiologist, who defined the organ edges on the CT images. To simplify the drawing process, preliminary volumes of interest (VOIs) for kidneys, liver, spleen, parotid glands, submandibular glands and urinary bladder were first drawn manually and then the “percent of maximum threshold” of an automatically adapted VOI in the corresponding PET images was varied, until the VOI matched the morphological delineated organ contour. The VOIs were then copied onto all other time points to generate the time-activity curves (TACs) for all segmented organs, respectively. A VOI covering the complete field of view was used as a surrogate to calculate the effective half-life in the total body, assuming that the distant limbs (cropped during PET/CT imaging) behave similarly as the trunk-torso. This effective half-life was then applied to the injected activity to derive the total body TAC. The TACs for red marrow were based on venous blood samples following established models , 14, excdecay T1/ 68 min sn OLINDA  using thn OLINDA .213Bi was used to calculate equivalent doses for all daughter nuclides  in OLINDA. The finally absorbed and effective doses are the sum of these nuclides, weighting 209Tl with 2% and 213Po with 98%, following the decay scheme of 213Bi  of alpha radiation with regard to deterministic effects was reviewed in detail by the US Department of Energy [213Bi. The uncertainties within this concept have already been discussed with regard to 225Ac-PSMA-617 [225Ac-PSMA-617 bench mark [The calculated absorbed doses were translated into equivalent doses using weighting factors of 5 for alpha and 1 for beta−/gamma radiation, from now on, referred to as “Svf Energy  and the f Energy , which cInterim results, such as residence time, biological and effective half-life, are provided in the Supplement/Table 225Ac-PSMA-617, which was based on surrogate imaging using 177Lu-PSMA-617 [Projected equivalent doses for salivary glands (which is not provided by OLINDA because they have not been considered in the Cristy–Eckerman phantom) and mean values for estimated tumor equivalent doses were calculated using the spherical model  already decayed by 98.9%, and 225Ac (T1/2 9.9 d) only decayed by 1.5%. Time-activity curves demonstrate how the half-life of the radiolabel and the pharmacokinetics of the shuttle molecule contribute to the tissue activity concentrations over time  was found sufficient to warrant clinical translation. Nevertheless, the therapeutic index seems clearly inferior compared to that of 225Ac-PSMA-617.The aim of this work was to estimate radiation dosimetry for 68Ga is a positron emitter and has the advantage of high resolution, overlap-free tomographic reconstruction and possible quantification by PET. As 68Ga has a minimally longer half-life than 213Bi, biodistribution data until near total decay of the planned therapeutics can be assessed, avoiding the common challenge that the integration to infinity has to be estimated by curve fitting. Nevertheless, the radio-metal in the chelator complex can theoretically affect the pharmacokinetics of the radiotracer and sometimes these effects are non-negligible [68Ga-PSMA-617 imaging to approximate 213Bi-PSMA-617 therapy presents both a major strength and simultaneously a mentionable limitation of the used methodology.It is common practice that the pharmacokinetics of therapeutic radiopharmaceuticals without sufficient inherent imaging capabilities are approximated by using a diagnostic surrogate radionuclide. gligible . Therefo213Bi-PSMA-617 has been extrapolated from PET data, but in contrast, the dosimetry of the comparator 225Ac-PSMA-617 has been estimated using planar scans—a necessary compromise to cope with its longer half-life. Due to the different physical properties of both radionuclides, the kidney-absorbed dose might be over-estimated for 213Bi-PSMA-617. At early time points, there is still activity in cavities of kidney calices/pelvis, contributing counts to the organ VOIs during imaging. But due to the limited penetration range of the alpha particles, activity deposits there would not reach the parenchyma in the renal cortex during TAT. In contrast, images of delayed time points in 225Ac-PSMA-617-dosimetry should include a higher fraction of activity representing specific uptake at renal PSMA or due to reabsorption by tubular cells and must be considered more toxically relevant.Another limitation is that the dosimetry of 225Ac-PSMA-617 contained remarkable methodical uncertainties because the limited resolution of the underlying planar scans hampers lesion delineation, contrast recovery and suffers from overlap, spill-out and partial volume effects. The a priori assumption that translocation of daughter nuclides can be neglected seems also more prudent for 213Bi than for the multi-step decay schema of 225Ac. However, the tumor to healthy organ ratios between 213Bi-PSMA-617 and 225Ac-PSMA-617 are in remarkably different dimensions


Lycosa erythrognatha is a potential model to develop new antibiotics against bacteria and fungi. In this work, we studied a peptide derived from LyeTxI, named LyeTxI-b, and characterized its structural profile and its in vitro and in vivo antimicrobial activities. Compared to LyeTxI, LyeTxI-b has an acetylated N-terminal and a deletion of a His residue, as structural modifications. The secondary structure of LyeTxI-b is a well-defined helical segment, from the second amino acid to the amidated C-terminal, with no clear partition between hydrophobic and hydrophilic faces. Moreover, LyeTxI-b shows a potent antimicrobial activity against Gram-positive and Gram-negative planktonic bacteria, being 10-fold more active than the native peptide against Escherichia coli. LyeTxI-b was also active in an in vivo model of septic arthritis, reducing the number of bacteria load, the migration of immune cells, the level of IL-1β cytokine and CXCL1 chemokine, as well as preventing cartilage damage. Our results show that LyeTxI-b is a potential therapeutic model for the development of new antibiotics against Gram-positive and Gram-negative bacteria.The antimicrobial peptide LyeTxI isolated from the venom of the spider  Super-resistant bacteria are an emerging public health problem. The urge to develop new tools and strategies to combat the superbugs is clear and was vehemently warned by the World Health Organization in a report about the global threat of antimicrobial resistance, in June 2014 . In a maAntimicrobial peptides are typically composed of 7–44 amino acid residues, usually exhibiting amphipathic alpha helix or beta-sheet conformations and being positively charged , althougLycosa erythrognatha. In particular, the venom peptides can interact with ion channels and cell receptors, and some of them show antimicrobial activity  and in vivo (a model of septic arthritis in mice), of LyeTxI-b, a peptide derived from LyeTxI. This peptide contains two structure modifications as acetylated N-terminal and a deletion of a His residue. These modifications have altered its structure and improved its activity in vitro and in vivo.Nowadays, several efforts have been made to design molecules derived from AMPs with better efficacy, low possibility to induce resistance and with less potential toxicity. Due to the great biotechnological/pharmaceutical potential of LyeTxI, its structure can be used as a template to develop new antibiotics against planktonic bacteria and fungi. Therefore, in the present work, we describe the structure and the biological activity, both 3CO-IWLTALKFLGKNLGKLAKQQLAKL-NH2), was synthesized by stepwise solid-phase using the N-9-fluorenylmethyloxycarbonyl (Fmoc) strategy (-1). The following side-chain protecting groups were used: t-butyl for threonine, t-butyloxycarbonyl for lysine and tryptophan, (triphenyl)methyl for asparagine and glutamine. The couplings were performed with 1,3-diiso-propylcarbodiimide (DIC), dichloromethane in DMF for 3–4 h. Fmoc deprotection steps were carried out with piperidine/DMF  . The last residue was deprotected and washed to perform the acetylation with a solution comprising 2 mL DMF, 1 mL DCM, 0.11 mol.L-1 DIC and 364.4 μl of 99% acetic anhydride (Sigma-Aldrich) for 40 min. The cleavage step and the side chains deprotection were performed with TFA/thioanisole/water/1,2-ethanedithiol/triisopropylsilane,  at room temperature during 3 h. The final product was precipitated with cold diisopropyl ether and lyophilized.The peptide LyeTxI-b (CHstrategy  on a Rin® BIO Wide Pore C18 column (Supelco), previously equilibrated with 0.1% aqueous TFA (solvent A). The elution was performed with a stepped gradient of acetonitrile in 0.1% TFA (solvent B) . The flow was 5.0 mL.min-1 and detection at 220 nm. The PepMap C18TM column (4.6 mm × 150 mm) previously equilibrated with solvent A was used in the second purification. The peptide fraction was eluted with a linear gradient of solvent B (0–100% of solvent B in 30 min). The flow was 1.0 mL.min-1 and detection at 214 nm.Two steps RP-HPLC were performed to purify the crude peptide. The first purification was done using a semi-preparative DiscoveryThe quality of peptide synthesis and purification were evaluated by MALDI-TOF/TOF mass spectrometry analyses carried out on an AutoFlex III instrument . The samples were co-crystallized with CHCA matrix  on MTP AnchorChip 400/384 or 600/384 plates . The instrument was operated in positive reflector mode and the results were analyzed on FlexAnalysis 3.1 .2O solutions , in the presence of SDS micelles (detergent concentrations ranging from 0.2 to 30.0 mM), and in the presence POPC:POPG (3:1 mol:mol) phospholipid vesicles (lipid concentrations ranging from 0.05 to 2.0 mM). The spectra were recorded at 20°C on a Jasco- J-735 spectropolarimeter coupled to a Peltier Jasco PTC-423L . A rectangular quartz cuvette  was used. Spectra were acquired from 260 to 190 nm using a 1.0 nm spectral bandwidth, 0.2 nm step resolution, 50 nm.min-1 scan speed, and 1 s response time. 6, 6, and 8 accumulations were, respectively, performed for the peptide samples prepared in TFE:H2O solutions, in the presence of detergent micelles and in the presence of phospholipid vesicles. Similar experiments with the respective blank solutions were also carried out for background subtraction. The peptide concentration, as determined from the tryptophan molar absorptivity , was at 36.5 nmol.L-1 in all CD studies. The POPC:POPG (3:1) phospholipid vesicles were prepared as described elsewhere  at 2.0 mM, and the pH was adjusted to 7.0 with 20 mM aqueous phosphate buffer. All NMR experiments were performed at 20°C on a Bruker Avance III spectrometer operating at a 1H frequency 600.043 MHz. A 5 mm triple-resonance (1H/13C/15N) gradient probe was used for all experiments. Water suppression was achieved by using pre-saturation. All NMR spectra were processed using NMRPipe  and Staphylococcus aureus (ATCC 25923) were cultured on BHI agar in aerobic conditions, while strains of Aggregatibacter actinomycetemcomitans (ATCC 29522) and Streptococcus sanguinis (ATCC 10556) were cultured on BHI in anaerobic conditions. The MIC using the microdilution method was performed to determine the bacterial susceptibility to AMPs, as previously described  values were determined by plating an aliquot of MIC values by well, with no visible turbidity, in BHI agar, in aerobic conditions for E. coli and S. aureus, and in anaerobic conditions for A. actinomycetemcomitans and S. sanguinis. After 24–48 h of growth, the MBC value was determined as the lowest concentration of peptide with no visible bacterial growth on the surface of the agar. MIC and MBC assays were performed in triplicate.Strains of escribed . Samples-1 to 0.17 μmol.L-1) for 1 h at 37°C. The released hemoglobin was measured using a spectrophotometry at 405 nm. Triton X-100% was used as positive control.The hemolytic assays were performed as previously described , with moStaphylococcus aureus ATCC 6538 was grown in BHI agar supplement with 5% of sheep blood for 24 h. The inoculum (107 CFU.mL-1) was injected into the tibiofemoral joint of anesthetized C57BL/6j mice as described  in 50 μL PBS emulsified in 50 μL Freund’s complete adjuvant . Fourteen days later, the antigen challenge was performed by injecting 10 μg mBSA  in the right knee joint of each mouse. Mice were then intra-articularly treated with LyeTxI-b (0.03 nmol) 1 h before the challenge and 3 h after the challenge. All procedures were performed under ketamine/xylazine anesthesia, and all efforts were made to minimize animal suffering. Analyses were conducted 24 h after the challenge. Non-immunized mice were used as negative controls. Euthanasia was performed as mentioned above.In vivo experiments were carried out in accordance and approved by the local Animal Experiment Ethics Committee .After mice euthanasia, the intra-articular cavity was exposed and washed with PBS containing 3% of bovine serum albumin (2× 5 μL). The total number of leukocytes and differential leucocytes was counted using a Neubauer chamber and cytospin preparations  stained with May–Grunwald–Giemsa, respectively. Then, the periarticular tissue was removed for cytokine analyses. IL-1β and CXCL1 chemokine concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA), following the instructions supplied by the manufacturer .In another set of experiments, mice were euthanized for the analysis of proteoglycan loss in joint cartilage, a marker of cartilage damage. Tissue was fixed in 10% buffered formalin (pH 7.4), decalcified for 30 days in 14% EDTA. Samples were embedded in paraffin and then sectioned and stained with toluidine blue. Two sections/knee joint were microscopically examined by a single pathologist to estimate joint proteoglycan content, as previously described . The joi® BIO Wide Pore C18 column  shows two major fractions eluted with an acetonitrile gradient (50–55% of solvent), named fraction A and fraction B. Only fraction B was submitted to another RP-HPLC experiment on a PepMap C18TM , and the relative purity of the fraction was analyzed by MALDI-TOF/TOF mass spectrometry. The peptide of interest was eluted in fraction B, which was evidenced by a higher m/z intensity 2736.4, corresponding to the expected mass of the N-terminally acetylated peptide (MW 2737.3 Da) . The peptide was therefore named LyeTxI-b. To confirm the primary structure of LyeTxI-b, this compound was subjected to MS/MS fragmentation .After chemical synthesis, we purified the peptide using two-step RP-HPLC. The first RP-HPLC assay on DiscoveryFigure 2A. Upon addition of TFE, the peptide adopts a helical conformation, as noticed by the appearance of two minima near 208 and 222 nm and a maximum near 192 nm , which show relatively small intensities up to 20% of TFE, however, significant increases are observed in solutions containing 30–50% of TFE, which present very similar spectral profiles. This is a very common feature of linear cationic AMPs peptides, which show no conformational preferences in aqueous environments, whereas well-defined conformations are observed in the presence of organic co-solvents or other membrane mimetic environments , the intensities of the characteristic maximum and minima increase with the SDS concentration until a plateau (5 mM of SDS) is reached. In the presence of vesicles , the minimum and maxima intensities also increase with the phospholipid concentration, however, significant augments in the two minima intensities are observed at 2 mM of phospholipids. These results indicate that the peptide adopts well-defined helical conformations in the presence of phospholipid vesicles, however, a membrane-bound  and a water-soluble (random coil) states coexist at smaller phospholipid to peptide ratios, as previously observed for other AMPs  vesicles and spectra with helical profiles were observed in both cases. In the presence of the micellar detergent  indicate a well-folded peptide conformation. Figure 4 presents the summary of through-space correlations that characterize peptide secondary structure, obtained from NOESY. In accordance with the data obtained from CD spectroscopy at similar conditions, several NN , αN , αβ  and αN  connections indicate that the peptide presents a well-defined helical segment from Trp-2 to the last amino acid residue, including the C-terminal carboxamide, which is involved in an αN  interaction. The distribution of distance and dihedral angle restraints per residue are presented in Figure 5A. This trend of α-helix secondary structure is also confirmed by the Hα and Cα chemical shift indexes (CSI) , indicating the good quality of the structural model obtained.Sequence-specific assignments have been performed from simultaneous analysis of NMR contour maps, as previously indicated . The dises (CSI) , as presFigure 3A), which is correlated to the NN  NOE correlations involving several residues of the helical segment. This sort of NOEs was observed for the native peptide with a minor proportion, inducing a less pronounced curvature of the peptide helix , since the positively charged side chain of this lysine residue interacts with a negatively charged aspartate residue and a hydrophilic serine residue of the other peptide chain. These electrostatic interactions are important to stabilize the homodimer coiled coil three-dimensional structure .Table 1). We observed that LyeTxI-b was exceptionally active against E. coli and S. aureus. The MBC values were the same as those obtained for MIC in all tests. The topic antibiotic chlorhexidine acetate was used as a positive control.LyeTxI-b showed a potent antimicrobial inhibition activity against Gram-positive and Gram-negative planktonic bacteria under aerobic and anaerobic conditions . It is noteworthy that in the growth inhibition tests we used 105 CFU.mL-1 while the SA model was established with 107 CFU.mL-1. Therefore, the MIC concentrations used was two-fold higher for the in vivo test. Thus, for each 10 μL injection in treatment models, 80 ng of LyeTxI-b and 3120 μg of clindamycin were applied.Considering our positive results regarding LyeTxI-b activity in the MIC assay, we evaluated the effectiveness of this peptide in a model of  in mice . InitialStaphylococcus aureus-induced joint inflammation is associated with a massive recruitment of neutrophils to the joint that, together with the presence of bacteria, cause articular damage and pain . Furthermore, LyeTxI-b reduces cell accumulation in S. aureus-challenged joint, including the number of total cells, neutrophils and mononuclear cells . Therefore, we can speculate that both treatments, using LyeTxI-b and clindamycin, were able to decrease the pain symptom, since the pain process can be associated with the mediators released by neutrophils . IL-1β has been shown as an important factor in the pathogenesis of septic arthritis in an experimental model of intravenous injection . Importantly, the injection of LyeTxI-b in naïve joint did not cause tissue inflammation.Similarly, the treatment with LyeTxI-b and clindamycin also reduced the level of IL-1β pro-inflammatory cytokine and CXCL1chemokine in the joint, when compared to non-treated joints . The treatment with LyeTxI-b did not alter the recruitment of neutrophils to the joint, which are important markers of tissue inflammation in this model . Thus, this result showed a non-direct role of LyeTxI-b on joint inflammation, and the reduction of S. aureus-induced arthritis seems to be caused by the control of bacterial growth in the joint. In sum, the ability of LyeTxI-b to control bacterial growth in the joint is sufficient to reduce the main inflammatory markers of SA model. In addition, preliminary nociceptive assays using an electronic version of von Frey test with LyeTxI-b in mice did not show any effect (data not shown).Our results show that LyeTxI-b-treated mice present decreased bacteria load and inflammation in the joint similar to clindamycin but in different concentration, as mentioned above. Since the presence of bacteria in a joint is sufficient to stimulate resident cells to produce pro-inflammatory mediators and cell recruitment, the reduced number of bacteria in LyeTxI-b-treated group could be enough to reduce all other inflammatory markers, as observed in Since the treatment of SA patients mostly involves use of antibiotics and considering that an increased number of patients infected with bacteria are resistant to antibiotics, the development of new therapeutic options became essential for a better control of infection and tissue damage . Thus, LIn the present work, we characterized LyeTxI-b, a peptide derived from the spider toxin LyeTxI, with two modifications: removal of a single amino acid residue and N-terminus acetylation. We show that these small alterations are able to improve the antibiotic activity of the peptide. This is also observed in the organisms under study, i.e., different strains of the same microorganism or different species may have different susceptibilities to the drug. Therefore, our results contribute to the emerging area of antibiotics derived from AMPs, as a new alternative for the treatment of infections that are resistant to conventional drugs.in vivo model. PR and MdL wrote the original draft. PR, MM-B, MC, AP, FA, JR, and MdL wrote, reviewed, and edited the manuscript. MdL envisioned the concept. DS, AP, and MdL supervised the study.PR synthesized and purified the peptide and performed MALDI-TOF and CD analyses. RV and JR performed the NMR experiments and analyzed the CD and NMR data. PR and MC investigated the study on MIC. PR and MM-B investigated the study on hemolysis. DB and FA investigated the study on The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Relinqing® granules (n = 6 per group). A metabolomics workflow including multivariate analysis and ROC regression was employed to select metabolic features that correlated with UTI and its treatment. Circa 4,000 molecular features were acquired for each sample. The small acid 3-hydroxybutyrate in plasma was found to be differentiated for urinary tract infection, with an area under the curve = 0.97 . The level of 3-hydroxybutyrate in plasma was depleted after infection with a fold change of -22 (q < 0.0001). Correlation between plasma 3-hydroxybutyrate and urine bacterial number in all groups and time points was r = -0.753 (p < 0.0001). The findings show that 3-hydroxybutyrate is depleted in blood and strongly associated with UTI at both infection and post-treatment stage in a UTI mouse model. Further work is envisaged to assess the clinical potential of blood tests to assist with UTI management.Urinary tract infection is one of the most common bacterial infections worldwide. Current diagnosis of urinary tract infection chiefly relies on its clinical presentation, urine dipstick tests and urine culture. Small molecules found in bio-fluids related with both infection and recovery would facilitate diagnosis and management of UTI. Mass spectrometry-based fingerprinting of plasma and urine at 3 time points, pre-infection (t = -24h), infection (t = 24h) and post 3-day treatment (t = 112h), were acquired in the following four groups: mice which were healthy, infected but not treated, infected and treated with ciprofloxacin, and infected and treated with  Escherichia coli (E.coli) which accounts for 80–85% of urinary tract infections  and correlation p(corr) values in the S-pot >0.60 and p[1]<-0.05, p(corr)<-0.80). Selected features were measured again in the raw data (area under the peak) and univariate analysis was performed. The receiver operating characteristic (ROC) curve was used to assess the prediction ability of the selected features. All statistical tests including t-test, Mann-Whitney test, Spearman correlation, and Wilcoxon signed ranks test were performed in SPSS . E.coli suspension were studied. It could be observed that with the increase of concentration, the urine bacterial number was also elevated. However, when the concentration reached 5×109 cfu/mL, the increase of bacterial number was not significant. The results are shown in The inoculation concentration and volume of 9 cfu/mL was chosen for the UTI mouse model, conditions that were in accordance with Lane et al [Inoculum volume experiments showed a similar pattern where the urine bacterial number increased moderately with the increase of inoculation volume , no bacterial colony was observed in the culture medium. At infection stage (T2), the urine culture results were positive for the mice infected with Plasma creatinine, a biomarker used routinely for kidney function, was also measured before and after infection . Result OPLS-DA model created from GC-MS plasma data revealed a significant variation between pre-infection (T1) and infection (T2) in terms of metabolic profiles . With thMetabolite semi-quantification: The peak areas of four metabolites were measured in the raw data  for univariate analysis. Lactate and cholesterol were excluded after univariate analysis as differences in the raw data were not significant.q < 0.001 and q < 0.001 respectively) and remained constant in the healthy control group (The result of paired t-test (two-tail) with multiple comparison correction indicated that 3-hydroxybutyrate (3-HB) and urea were significantly different after infection in infected group  analysis. Creatinine, the indicator for kidney failure, was also fitted in the analysis to provide a reference. The area under curve (AUC) for 3-HB and urea were 0.97  and 0.85 , respectively. The combination of 3-HB and urea produced an AUC of 0.98, but the sensitivity decreased. Therefore, data for 3-HB only were included for further analysis. For comparison, the AUC for creatinine was 0.51  ( = 0.13) .Similar data analysis approaches were applied to urine GC-MS data between pre-infection (T1) and infection (T2) stages in infected group. Five features were found to contribute most for group separation. After comparison with the database, they were putatively identified as pyroglutamate, meso-Erythritol and L-(-)-Arabitol, deoxyglucose and D-(+)-Talose.Metabolite semi-quantification: The peak areas of these metabolites were measured in the raw data  for univariate analysis. The result of paired t-test (two-tail) with multiple comparison correction demonstrated that among them pyroglutamate, meso-Erythritol and L-(-)-Arabitol were elevated greatly after infected with E.coli in infected group (q < 0.01) and did not change in the healthy control group . p value < 0.0001 (for RLQ and CPF treatments it was -0.76 and -0.72 respectively).The level of 3-HB in plasma from T1 to T3 was measured. A line graph based on time points was drawn from its semi-quantification data to depict changes at three time points . The quaThe concentration of urine 3-HB also decreased greatly at infection stage in the infected group. In the healthy control group, mice at T1 and T2 showed a similar level of 3-HB . A line L-(-)-Arabitol and meso-Erythritol were not detectable in plasma, while the amount of pyroglutamate showed no significant difference between pre- and post-infection (data not shown).The levels of urine pyroglutamate, meso-Erythritol and L-(-)-Arabitol from pre-infection (T1) to post-treatment (T3) were also measured to investigate any potential of reflecting the infection recovery process. These metabolites, instead of following a clear pattern like plasma 3-hydroxybutyrate, showed changes that were not significant overall. Results are shown in The aforementioned findings suggested that 3-hydroxybutyrate might be a candidate metabolite for UTI detection and its management. A pure reference standard of 3-hydroxybutyrate was analysed to confirm the identity of 3-hydroxybutyrate in the samples .E.coli-infected mice. For the UTI model study, the inoculation concentration and volume were investigated. Plasma culture was used to assess if there was dissemination of bacteria into the blood during infection and plasma creatinine was measured to evaluate kidney function. Before infection, urine samples were collected and cultured to examine if the mice urine was normal. At T1  stage no bacterial colony was observed in the culture medium which demonstrated that the urine samples were not contaminated and all the mice were free from bacteria before infection.In our study, we investigated the mouse metabolic response to urinary tract infection with GC-MS-based metabolomics. A significant correlation between 3-HB in plasma and urinary bacterial number/UTI was observed, showing a 22-fold reduction of 3-HB levels in the plasma of To provide an estimate of the prediction ability of selected metabolites, ROC analysis was performed. In ROC curve analysis, sensitivity estimates how good the test is in identifying a disease while specificity evaluates how likely a subject without disease can be ruled out by the test. AUC gives the overall accuracy of the test and can be used to compare different test performances . In plasUrine pyroglutamate, meso-Erythritol and L-(-)-Arabitol combined returned an AUC of 0.83. This suggested a predictive potential of more than 80%, however the sensitivity was only 0.75 and their metabolic variation did not correlate with the recovery process.Relinqing® granules, the exact mechanism of action is still unknown. The level of 3-HB increased after the two different treatments, indicating that 3-hydroxybutyrate should be related to infection and the recovery. We observed a slower bacteriostatic effect with the RLQ treatment compared with CPF during the three-day period. This was reflected in both the urine bacterial number and plasma 3-HB levels. The correlation of 3-HB to bacterial number in RLQ and CPF treatments was -0.76 and -0.72 respectively.After treatment (T3), the levels of 3-HB rebounded in plasma in both CPF and RLQ groups while 3-HB remained unchanged in the UTI group. However, in urine, the level of 3HB didn’t go back. It is because the excretion of 3HB involves renal ultrafiltration, reabsorption and secretion. All of these could make the urine profile show a slower recovery process. By including two different treatments in this experiment, we wanted to increase the reliability of any UTI-related metabolites in plasma. The first treatment, ciprofloxacin, kills bacteria by inhibiting its DNA gyrase  and the This small acid 3-hydroxybutyrate is one of three ketone bodies together with acetoacetate and acetone. Ketone bodies are generated from β-oxidation of free fatty acids in liver mitochondria  with thrE.coli, is a toxin that is released by E.coli. It can induce the secretion of pro-inflammatory cytokines [Lipopolysaccharide, a major component of the outer membrane of ytokines . LPS andytokines , 44. Theet al. and Wannemacher et al. observed a decreased concentration of free fatty acid in plasma during infection [Moreover, the inner mitochondrial membrane is impermeable for fatty acyl-CoA, it demands a special carrier system to transfer fatty acyl-CoA to the mitochondrial matrix shown in nfection –49.Plasma metabolic phenotyping is regarded as a collective "snapshot" of changes within the body's metabolism, thus the alteration of plasma metabolites could serve as good indicators of health or disease status . The resE.coli-infected group and ROC curve revealed that 3-HB had a prediction ability of 0.97 (95% CI: 0.93–1.00). A targeted longitudinal study of plasma 3-HB was also monitored which revealed that 3-HB was able correlate with the recovery process. In addition, the reduced 3-HB was strongly associated with urine bacterial number . Further investigations will focus on other bacterial strains in UTI.Our results indicated that 3-HB in plasma highly correlated to urinary tract infection with a specificity of 0.91 and sensitivity of 0.92. It showed a significant decrease in the S1 FigRelinqing®granules.UTI: mice treated with saline; CPF: mice treated with ciprofloxacin; RLQ: mice treated with (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigA. The change of urine pyroglutamate concentration in different groups at different time points (n = 6 per group at each time point). B. The change of urine meso-Erythritol in different groups at different time points (n = 6 per group at each time point). C. The change of urine L-(-)-Arabitol concentration in different groups at different time points (n = 6 per group at each time point).(TIF)Click here for additional data file.S4 FigA. Chromatogram and mass spectrum of 3-hydroxybutyrate reference standard. B. Chromatogram and mass spectrum of 3-hydroxybutyrate from a plasma sample.(TIF)Click here for additional data file.S5 FigThe enzyme highlighted in yellow is expressed differently in mouse and human.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Agrobacterium mediated transformation. Stable expression of SP-LL-37 in transgenic rice plants was confirmed by RT-PCR and ELISA analyses. Subcellular localization of SP-LL-37-GFP fusion protein showed evidently in intercellular space. Our data on testing for resistance to bacterial leaf blight and blast revealed that the transgenic lines are highly resistant compared to its wildtype. Our results suggest that LL-37 can be further explored to improve wide-spectrum resistance to biotic stress in rice.Human LL-37 is a multifunctional antimicrobial peptide of cathelicidin family. It has been shown in recent studies that it can serve as a host’s defense against influenza A virus. We now demonstrate in this study how signal peptide LL-37 (SP-LL-37) can be used in rice resistance against bacterial leaf blight and blast. We synthesized LL-37 peptide and subcloned in a recombinant pPZP vector with pGD1 as promoter. SP-LL-37 was introduced into rice plants by  As rice is the main food source for more than half of the world's population, fluctuation in production can indirectly make a significant impact on the world economy. Bacterial leaf blight and blast are the most important diseases affecting rice production. They are difficult to control because the speed of diffusion is very fast. The usual way to control such type of pathogens is to spray pesticides. However, continuous and extensive use of such pesticides causes long-term exposure of the environment to contamination. In addition, many microorganisms can acquire resistance to chemical pesticides . The mosAntimicrobial peptides (AMPs) are components of the defense system of animals and plants against pathogens and are generally shorter than 50 amino acid residues per peptide. AMPs are controlled by a single gene, and can destroy microorganisms, including bacteria, fungi, mycoplasma, and viruses, with minimum energy consumption during the initial infection. Because of this huge advantage, scientists used AMPs as one of the important sources for breeding resistant varieties . The curHyalophora cecropia Linnaeus in 1980 and they were considered as AMPs derived from animals [Erwinia carotovora and Pseudomonas syringae pv. tabaci [Cecropins were isolated from the silkworm moth  animals . Rice tr animals . Express. tabaci –7. LaterAiming for production of a mature peptide using signal sequences of different origin has achieved a relatively high success rate , 9. CECMXanthomonas oryzae in leaves that cause leaf blight disease. Transgenic plants also showed high resistance against blast disease caused by Magnaporthe oryzae. The SP-LL-37 transgenic rice will be a valuable breeding line for resistant variety development.LL-37 is the only member of the cathelicidin family of antimicrobial peptides isolated from humans. It is an amphipathic, 37-residue-long ɑ-helical peptide which has a broad spectrum of antibacterial activity . The intOryza sativa L. var. Japonica cv. Dongjinbyeo  was used in plant transformation. There were 472 T0 plants used for PCR analysis to confirm the transformation.Rice cultivar The wild type Dongjinbyeo and transgenic lines were grown in paddy field condition. At maturity, agronomic traits were evaluated for chlorophyll content, days to flowering, plant height, culm length (CL), panicle length (PL), panicles per plant (PPP), spikelets per panicle (SPP), percent ripened grain (PRG), and 1000-grain weight (TGW). Evaluation methods were similar to that described in Cho et al. (2007) .KpnI and SmaI enzyme sites. The whole sequence of LL-37 is shown in Agrobacterium (A. tumefaciens strain EHA105) mediated transformation. Transgenic plants were grown in 1/2 MS (Murashige-Skoog) plates supplemented with 6 mg/L PPT to screen for PPT-resistant lines before cultivation in soil. Introduction of the gene in transgenic plants was confirmed by PCR using the primers 5’-ATGTTGTTGGCAATTGCTTTCC-3’ (Forward) and 5’- CTAGGACTCTGTCCTGGGTACAAG-3’ (Reverse) for the SP-LL-37 gene and all other primer sequences used for the subsequent experiments are listed in The recombinant binary pPZP vector was constructed by inserting SP-LL-37 synthetic gene in the TaqMan real-time quantitative PCR was performed with a LightCycler® 480 machine and the LightCycler® 480 Probes Master kit (Roche). Reactions have a final volume of 10 μL composing of 1× LightCycler 480 Probes Master, 0.5 μM forward primer, 0.5 μM reverse primer, 0.2 μM TaqMan probes and 2.5 μL of the DNA template. Amplification conditions were as follows: denaturation at 95℃ for 10 min, followed by 50 cycles of amplification  and cooling at 40℃ for 10 sec. All samples were tested through qPCR for both 5' and 3' regions of the ribosomal RNA gene for selection of single copy transgenic plants, the method used in this study was described by Wang et al .0 transgenic plants were used to selected single copy transgenic plants and later used for inverse PCR adjacent sequencing of the inserted T-DNA. Using the FSTs (flanking sequence tags), the estimated position at which the T-DNA was introduced was determined after matching through blast program. IPCR (inverse PCR) is a method that depends on the results of digestion of the genome near the inserted T-DNA, using a specific restriction enzyme. Since the whole genome sequences of rice digested with BfaI have around 500 bp gaps, an experiment was designed to check this restriction enzyme. To increase the recovery efficiency of FSTs, the FSTs reaction was performed in both left border (LB) and right border (RB) of the T-DNA. Primers suitable for amplification of the T-DNA from the FSTs analysis were made after testing the efficiency of each LB and RB associated primer based on the BfaI adapter primer , after which the T4 DNA ligase was inactivated by incubating the samples at 65℃ for 10 min. Next, 7 μL of adapter-ligated genomic DNA was transferred to a fresh tube/well, followed by addition of 43 μL of a mixture containing H2O, Taq DNA polymerase, buffer, dNTPs and primers. To amplify the regions flanking the RB of the T-DNA insert, AP1 and Fa1 or L1 primers were used. For amplification of the regions flanking the LB of the T-DNA insert, the AP1 and T-DNA primers were employed (Ten μL of plant genomic DNA (up to 100 ng) was added to a fresh tube. The genomic DNA was then digested with employed , Fig 2A.Agrobacterium mediated injection. Young apical leaves (5 ~ 6 leaves per plant) were infiltrated with recombinant Agrobacterium strains using a syringe (2 mL). Leaves were superficially wounded with a needle to improve infiltration. For constructs pGFPt and LL-37::GFP, a single zone on the opposite half of the leaves of N. tobaccum was infiltrated. The Agrobacterium strain containing pGFPt was used as a control. Three plants were agroinfiltrated at each time, and each experiment was repeated twice. The agroinfiltrated leaves were photographed at 36 ~ 48 h following infiltration.By fusing the C-terminus of SP-LL-37 and GFP gene, a pGWB5::SP-LL-37 vector was constructed. Tobacco leaf was transformed with pGWB5::SP-LL-37 using The visual detection of GFP fluorescence was performed on agroinfiltrated leaves. The transformed leaves were photographed using a confocal Olympus FV500 equipped with a combined laser with an excitation of 488 nm and emission of 520 nm .In planta bacterial leaf blight assay was carried out using 20 plants each from T2 generation, as well as untransformed control plants, using ~1×107 cells ml-1 of X. oryzae pv. oryzae following the ‘scissors clip-inoculation’ method [’ method . A pair To test the resistance of transgenic plants to rice blast, artificial inoculation and field evaluations were carried out. At four-leaf stage, seedlings were inoculated by spraying with a spore suspension of fungal strain in 0.02% Tween-20 with a concentration of 100 spores in 100× visual field. After inoculation, the seedlings were kept at 26 ~ 28°C with saturated humidity for 24 h in the dark, and then transferred to an artificial climate-tunnel at 26°C. Disease reactions were scored about 7 days after inoculation using the standard described by Pan et al., 1996 .M. oryzae strain was selected because it causes partiality to Dongjinbyeo. Spores were obtained by treatment under a fluorescent lamp at 28°C for 48 hours, and then precipitated in sterile water after 7 days of incubation in oatmeal medium. The spore concentration of 10*10 per 100 x field was determined using a microscope and the spores were suspended with a concentration of 105 CFU / ml. Spore suspension was made by addition of 0.2% Tween 20. Seedlings of at four-leaf stage were then inoculated by manual spraying.et al., 2004 [2O2 were added. After incubation, the reaction was measured at 405 nm with an ELISA reader.Protein extraction and analysis of LL-37 expressing lines were performed as described by Alan l., 2004 . The totAgrobacterium-mediated method. 472 (40%) transgenic plants from a total of 1,180 PPT resistant calli were regenerated  analysis using specific primers for BfaI adapter and T-DNA primers flanking the LB and RB sites :yellow (1) ratio. To select homo lines, a NOS gene probe was used in Taqman PCR for copy number analysis. The transgenic lines 6–1, 6–3, 12–1, 12–2, 12–3, 16–3, 17–3, 17–4, and 20–1 were all identified to have one copy number, and were therefore selected as homo lines . Total RWhen developing varieties producing antimicrobial peptides, stable expression must be considered. Since antimicrobial peptides are very short, they are susceptible to degradation by the proteases of the plant. LL-37 expression was maximized by using a signal peptide targeting it for secretion into the intercellular space, where it would have the least impact on the plants. To test whether the signal peptide indeed induced secretion of LL-37 from the nucleus to the intercellular space, subcellular localization by tobacco transient assay was performed. SP-LL-37::GFP fusion protein and a GFP only constructs  were injX. oryzae KACC 10859 strain (K3a)  . The K3a in size .In addition, to see if LL-37 has a broad spectrum of resistance, we inoculated the transgenic lines with blast fungus. Seven days after inoculation of rice blast strain, the percent frequency of diseased leaf area (DLA) was measured. Visually striking yellowish symptoms began to appear bordering the spindle in WT plants. From 14 days onward, the symptoms had spread throughout the leaf and were clear enough to distinguish the transgenic lines . ApproxiThe transgenic SP-LL-37 homo lines obtained in this work were compared with WT in terms of agronomic traits . No sign0 generation in order to ensure a stable expression.Human cathelicidin antimicrobial protein LL-37 has attracted much attention because of its potential for broad spectrum antimicrobial activity, and the fact that it can be used as a substitute for antibiotics in the development of transgenic rice. We have already developed homozygous Chinese cabbage and tomato lines that stably expressed LL-37, which were found to be resistant to bacteria and fungi , 21. FurBacterial leaf blight inoculation experiments on four lines revealed significantly higher disease resistance than WT. The four transgenic lines showed lesions from 0.7 cm to 2.2 cm in size, which were smaller than the lesion length of 10.8 cm in WT. The transgenic plants did not show more signs of disease progression. In rice blast experiment, fusiform lesions were appeared as dark brown areas in over 73.2% of the control leaves, while substantial growth was not observed on the transgenic lines, which displayed lesions covering from 3.1 to 9.0% of the area. Therefore, the LL-37 gene product in the transgenic lines showed antibacterial activity against bacterial leaf blight and rice blast. In a different study, LL-37 has been reported to increase the expression of the PR-related protein in transgenic tomato, it increased the expression of defense-related gene response .The expression of antimicrobial peptides in plants often fails due to the influence of proteases. For example, the Cecropin B peptide has a short persistence with the half-life of 25.5 h due to the effect of protease in transgenic rice . To prevPlant development for enhancing disease resistance is an important area to eliminate the damage caused by the widespread use of chemical pesticides. In this study, we showed how to minimize destruction to the rice genome by selecting single, intergenic lines among the SP-LL-37 transgenic lines. Furthermore, transgenic lines with SP-LL-37 showed enhanced resistance to bacterial leaf blight and blast fungus, achieved through stable expression and localization in the intercellular area using a signal peptide. The SP-LL-37 transgenic rice, line 17–4, should prove valuable to the use of AMPs to generate crops with wide spectrum of resistance to any disease or harmful environment.S1 Table(DOCX)Click here for additional data file.